Use of Saccharomyces cerevisiae erg4 mutants for expressing mammalian glucose transporters

ABSTRACT

The invention relates to yeast strains in which a human GLUT4 transport or a human GLUT1 transporter can be functionally expressed and to particular GLUT4 transport proteins which can be functionally expressed particularly readily in yeast strains.

This application is a continuation of U.S. patent application Ser. No.11/775,384, filed on Jul. 10, 2007, which issued as U.S. Pat. No.7,615,360 on Nov. 10, 2009; which is a continuation of U.S. patentapplication Ser. No. 10/659,234, filed on Sep. 10, 2003, which issued asU.S. Pat. No. 7,244,821 on Jul. 17, 2007; which claims priority fromU.S. Provisional Patent Application No. 60/455,340, filed on Mar. 17,2003, now expired, and German Patent Application No. 10242763.1, filedon Sep. 14, 2002, each of which is incorporated, herein, in theirentireties.

The invention relates to yeast strains in which the human Glut 4 andGlut 1 transporters can be functionally expressed.

Most heterotropic cells transport glucose via special transporterproteins into the cell interior. The various organisms have developeddifferent mechanisms mediating the transporting of glucose, such as, inparticular, proton symport systems, Na⁺ glucose transporters, bindingprotein-dependent systems, phosphotransferase systems, and systems forfacilitated diffusion. In the eukaryotes, a family of glucosetransporters which are encoded in mammals by the GLUT genes(GLUT=glucose transporter) and Saccharomyces cerevisiae by the HXT genes(HXT=hexose transporter) mediates glucose uptake via facilitateddiffusion. Said transporters belong to a larger family of sugartransporters. They are characterized by the presence of 12 transmembranehelices and by a plurality of conserved amino acid radicals. Glucosetransport plays an important part in disorders associated with adefective glucose homeostasis, such as, for example, diabetes mellitusor Fanconi-Bickel syndrome. The glucose transport in mammals hastherefore been the subject of numerous studies. To date, thirteenglucose transporter-like proteins have been identified (GLUT1 to GLUT12,HMIT—H-myo-inositol transporter)). Said transporters play key partswhich include the uptake of glucose into various tissues, its storage inthe liver, its insulin-dependent uptake into muscle cells and adipocytesand glucose measurement by the β cells of the pancreas.

GLUT1 mediates the transport of glucose into erythrocytes and throughthe blood-brain barrier, but is also expressed in many other tissues,while GLUT4 is limited to insulin-dependent tissues, primarily to muscleand fatty tissue. In said insulin-dependent tissues, controlling thetargeting of GLUT4 transporters through intracellular compartments orplasma membrane compartments represents an important mechanism forregulating glucose uptake. In the presence of insulin, intracellularGLUT 4 is redistributed through the plasma membrane in order tofacilitate glucose uptake. GLUT1 is likewise expressed in saidinsulin-dependent tissues, and its distribution in the cell is likewiseinfluenced by insulin, albeit not as strongly. In addition, the relativeefficacy with which GLUT1 or GLUT4 catalyze sugar transport isdetermined not only by the extent of the targeting of each transporterto the cell surface but also by their kinetic properties.

The fact that different glucose transporter isoforms are coexpressed andthe rapid glucose metabolism have rendered studies on the role and theexact properties of each glucose transporter isoform in theseinsulin-dependent tissues complicated. In order to solve these problems,heterologous expression systems such as Xenopus oocytes, tissue culturecells, insect cells and yeast cells have been used. However, it turnedout that a number of difficulties appeared in connection with thesesystems: too weak an activity of the heterologously expressedtransporters, intrinsic glucose transporters in said systems,intracellular retention of a considerable proportion of the transportersor even production of inactive transporters.

Naturally occurring GLUT4 protein of mammals, in particular that ofhumans, can be expressed in a functional manner in strains ofSaccharomyces cerevisiae under particular conditions.

Yeast cells are unicell eukaryotic organisms. They are therefore, forsome proteins, more suitable for expression than bacterial systems, inparticular with regard to carrying out screen assays for identifyingpharmaceutically active substances.

The present invention relates to a purified and isolated polynucleotidecomprising a DNA sequence which codes for the GLUT4V85M protein.

Said protein contains at position 85 of the amino acid chain of thehuman GLUT4 protein an amino acid exchange from valine to methionine.This altered GLUT4V85M protein provides further alternatives forexpressing a functional GLUT4 protein. A GLUT4 protein should beregarded as functional in connection with Saccharomyces cerevisiae ifglucose uptake can be observed in a Saccharomyces cerevisiae strainwhose glucose transporters in their entirety are inactive (=hxt(−))after expression of said GLUT4 protein. Glucose uptake may be determinedeither by transport measurements by means of radioactively labeledglucose or by growth on medium with glucose as sole carbon source.

In a preferred embodiment, the purified and isolated polynucleotidecomprising a DNA sequence which calls for a protein GLUT4V85M mayinclude or comprise a sequence of the following groups:

-   -   a) a nucleotide sequence according to Seq ID No. 1,    -   b) a nucleotide sequence which hybridizes to a sequence of Seq        ID No. 1 under stringent conditions and which codes for a        protein GLUT4V85M.

The purified and isolated polynucleotide preferably encodes a GLUT4V85Mprotein which has an amino acid sequence of Seq ID No. 2.

The purified and isolated polynucleotide comprising a DNA sequence whichcodes as discussed previously for a protein GLUT4V85M, may beoperationally linked to a promotor. Suitable promotors are in particularprokaryotic or eukaryotic promoters such as, for example, the Lac-,trp-, ADH- or HXT7 promotor. The part of the polynucleotides, whichcodes for the protein GLUT4V85M is operationally linked to a promotorprecisely if a bacterial or eukaryotic organism produces, by means ofsaid promotor with the aid of a vector, an mRNA which can be translatedinto the protein GLUT4V85M. An example of such a vector is the vectorp4H7GLUT4V85M (Seq ID No. 3). The protein GLUT4V85M may be expressed inyeast cells by means of said vector.

The above-described polynucleotide comprising a DNA sequence which codesfor a protein GLUT4V85M is, in a preferred embodiment, suitable forreplicating said polynucleotide in a yeast cell or for expressing thepart of the polynucleotide, which encodes the protein GLUT4V85M, in ayeast cell to give the protein GLUT4V85M. A yeast cell fromSaccharomyces cerevisiae is particularly suitable. For replication andexpression in a yeast cell, the polynucleotide comprising a DNA sequencewhich calls for a protein GLUT4V85M is present in the form of a yeastvector. The polynucleotide region coding for the GLUT4V85M protein maybe operationally linked to a yeast cell-specific promotor such as, forexample, the ADH promotor (alcohol dehydrogenase promotor) or the HXT7promotor (hexose-transporter promotor). The yeast sectors are a group ofvectors which was developed for cloning of DNA in yeasts.

The invention furthermore relates to a yeast cell from Saccharomycescerevisiae in which all glucose transporters are no longer functional(=hxt (−)) and which contains no functional Erg4 protein. Such a yeastcell is preferably a yeast cell deposited as Saccharomyces cerevisiaeDSM 15187 with the DSMZ (Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH, Mascheroder Weg 16, 38124 Brunswick, Germany).

The invention also relates to a yeast cell in which all glucosetransporters are no longer functional and which contains no functionalFgy1 and no functional Erg4 protein. The lack of an Erg4 protein or ofan Fgy1 protein may be attributed in particular to an interruption ofthe corresponding coding genome sections or to a partial or completeremoval of said coding genome sections.

Preference is given to using as yeast cell which contains no functionalglucose transporters, no functional Fgy1 protein and no functional Erg4protein, a yeast cell as deposited with the DSMZ as Saccharomycescerevisiae DSM 15184.

A yeast cell as described above is preferably used for expressing amammalian GLUT1 protein or a mammalian GLUT4 protein, in particular aprotein from rats, mice, rabbits, pigs, cattle or primates. A preferredembodiment uses the yeast cell for expressing a human GLUT4 or GLUT1protein.

A Saccharomyces cerevisiae yeast cell whose glucose transporters intheir entirety and also the Erg4 protein are no longer functional maycontain a polynucleotide of the present invention, which comprises a DNAsequence coding for a protein GLUT4V85M. Said yeast cell can alsoexpress the GLUT4V85M protein and thus contain said protein.

A yeast strain of this kind, containing a polynucleotide which comprisesa DNA sequence coding for the GLUT4V85M protein, is preferably theSaccharomyces cerevisiae DSM 15185 yeast strain which has been depositedwith the DMSZ.

A yeast cell whose glucose transporters in their entirety and also theErg4 protein are no longer functional and which contains apolynucleotide comprising a DNA sequence which calls for a proteinGLUT4V85M may be prepared, for example, by

-   -   a) providing a yeast cell whose glucose transporters in their        entirety and also the Erg4 protein are no longer functional,    -   b) providing an isolated and purified polynucleotide which        comprises a DNA sequence coding for the GLUT4V85M protein and        which can be replicated in the yeast cell,    -   c) transforming the yeast cell from a) with the polynucleotide        from b),    -   d) selecting a transformed yeast cell,    -   e) where appropriate expressing the GLUT4V85M protein.

An isolated and purified polynucleotide which comprises a DNA sequencecoding for the GLUT4V85M protein is preferably a vector which can bereplicated in a yeast cell and in which said DNA sequence was cloned. Anexample of such a vector is p4H7GLUT4V85M (Seq ID No. 3).

The invention also relates to a yeast cell whose glucose transporters intheir entirety and whose proteins for Fgy1 and Erg4 are no longerfunctional and which contains a polynucleotide which comprises a DNAsequence coding for the GLUT4V85M protein. Said yeast cell can alsoexpress the GLUT4V85M protein and thus contain said protein. A yeaststrain of this kind is preferably the Saccharomyces cerevisiae DSM 15186deposited with the DSMZ.

A yeast cell whose glucose transporters in their entirety and also theproteins Fgy1 and Erg4 are no longer functional and which contains apolynucleotide comprising a DNA sequence which codes for the GLUT4V85Mprotein may be prepared, for example, by

-   -   a) providing a yeast cell whose glucose transporters in their        entirety and also the proteins Fgy1 and Erg4 are no longer        functional,    -   b) providing an isolated and purified polynucleotide which        comprises a DNA sequence coding for the GLUT4V85M protein and        which can be replicated in the yeast cell,    -   c) transforming the yeast cell from a) with the polynucleotide        from b),    -   d) selecting a transformed yeast cell,    -   e) where appropriate expressing the GLUT4V85M protein.

The abovementioned isolated and purified polynucleotide which comprisesa DNA sequence coding for the GLUT4V85M protein is preferably a vectorwhich can be replicated in a yeast cell and in which said DNA sequencewas cloned. An example of such a vector is p4H7GLUT4V85M (Seq ID No. 3).

The invention also relates to a yeast cell whose glucose transporters intheir entirety are no longer functional and which contains apolynucleotide comprising a DNA sequence which calls for the GLUT4V85Mprotein.

Said yeast cell can also express the GLUT4V85M protein and thus containsaid protein. A preferred yeast strain of this kind is the Saccharomycescerevisiae 15188 yeast strain deposited with the DSMZ.

A yeast cell whose glucose transporters in their entirety are no longerfunctional and which contains a polynucleotide comprising a DNA sequencewhich codes for the GLUT4V85M protein may be prepared, for example, by

-   -   a) providing a yeast cell whose glucose transporters in their        entirety are no longer functional,    -   b) providing an isolated and purified polynucleotide which        comprises a DNA sequence coding for the GLUT4V85M protein and        which can be replicated in the yeast cell,    -   c) transforming the yeast cell from a) with the polynucleotide        from b),    -   d) selecting a transformed yeast cell,    -   e) where appropriate expressing the GLUT4V85M protein.

An isolated and purified polynucleotide which comprises a DNA sequencecoding for the GLUT4V85M protein is preferably a vector which can bereplicated in a yeast cell and in which said DNA sequence was cloned. Anexample of such a vector is p4H7GLUT4V85M (Seq ID No. 3).

The invention also relates to a protein having the amino acid sequenceaccording to Seq ID No. 2. Said protein is a human GLUT4 protein inwhich a valine has been replaced by a methionine in position 85 of theamino acid chain.

The invention also relates to a method for identifying a compound whichstimulates the activity of a GLUT4 protein, which method comprises thesteps

-   -   a) providing a yeast cell whose glucose transporters in their        entirety and also Erg4 protein are no longer functional and        which contains a polynucleotide comprising a DNA sequence which        codes for a protein GLUT4V85M,    -   b) providing a chemical compound,    -   c) contacting the yeast of a) with the chemical compound of b),    -   d) determining glucose uptake by the yeast of c),    -   e) relating the detected value of the glucose uptake of d) to        the detected value of glucose uptake in a yeast cell as claimed        in a) which has been contacted with a chemical compound as        claimed in b), with a compound which causes an increase in the        amount of glucose taken up in the yeast as claimed in d)        stimulating the activity of said GLUT4 protein. Compounds which        stimulate the activity of the GLUT4V85M protein can be assumed        to stimulate also the GLUT4 activity.

The invention also relates to a pharmaceutical which contains a compoundwhich has been identified by the method described above and furthermoreto additives and excipients for formulating a pharmaceutical.Furthermore, the invention relates to the use of a compound which hasbeen identified by the method described above for producing apharmaceutical for the treatment of type I and/or II diabetes.

The invention also relates to a pharmaceutical comprising a compoundwhich has been identified by the method described above and to additivesand excipients for formulating a pharmaceutical. Furthermore, theinvention relates to the use of a compound identified by the methoddescribed above for producing a pharmaceutical for the treatment ofdiabetes.

The invention furthermore relates to the use of a compound identified bya method described above for producing a pharmaceutical for thetreatment of diabetes.

The present invention also comprises a method for identifying a compoundwhich inhibits the protein encoded by the Erg4 gene, which methodcomprises the steps:

-   -   a) providing a yeast cell whose glucose transporters in their        entirety and no longer functional and which contains a        polynucleotide comprising a DNA sequence which codes for the        GLUT4V85M protein and can be replicated in a yeast cell,    -   b) providing a chemical compound    -   c) contacting the yeast of a) with the chemical compound of b),    -   d) determining glucose uptake by the yeast of c),    -   e) relating the detected value of the glucose uptake of d) to        the detected value of glucose uptake in a yeast cell as claimed        in a) which is not contacted with a chemical compound as claimed        in b), with a compound which causes an increase in the amount of        glucose taken up in the yeast as claimed in d) stimulating the        activity of a protein Erg4.

The invention furthermore relates to a method for identifying a compoundinhibiting the corresponding protein of the Fgy1 gene, which comprisesthe steps:

-   -   a) providing a yeast cell whose glucose transporters in their        entirety and whose Erg4 protein are no longer functional and        which contains a GLUT4 protein,    -   b) providing a chemical compound    -   c) contacting the yeast of a) with the chemical compound of b),    -   d) determining glucose uptake by the yeast of c),    -   e) relating the detected value of the glucose uptake of d) to        the detected value of glucose uptake in a yeast cell as claimed        in a) which is not contacted with a chemical compound as claimed        in b), with a compound which causes an increase in the amount of        glucose taken up in the yeast as claimed in d) stimulating the        activity of a protein Fgy1.

The invention also relates to a pharmaceutical comprising a compoundwhich has been identified by the method described above and to additivesand excipients for formulating a pharmaceutical.

The invention may be illustrated in more detail below with respect totechnical details.

Hybridization means the assembling of two nucleic acid single strandshaving complementary base sequences to double strands. Hybridization maytake place between two DNA strands, one DNA and one RNA strand andbetween two RNA strands. In principle, it is possible to prepare hybridmolecules by heating the nucleic acids involved which may initially bein double-stranded form, by boiling, for example, in a waterbath for 10minutes, until they disintegrate into single-stranded molecules withoutsecondary structure. Subsequently, they can be cooled slowly. During thecooling phase, complementary chains pair to give double-stranded hybridmolecules. Under laboratory conditions, hybridizations are usuallycarried out with the aid of hybridization filters to whichsingle-stranded or denaturable polynucleotide molecules are applied byblotting or electrophoresis. It is possible to visualize thehybridization using appropriate complementary polynucleotide moleculesby providing said polynucleotide molecules to be hybridized with aradioactive fluorescent label. Stringency describes the degree ofmatching or alignment of particular conditions. High stringency hashigher demands on matching than low stringency. Depending on theapplication and objective, particular conditions with differentstringency are set for the hybridization of nucleic acids. At highstringency, the reaction conditions for the hybridization are set insuch a way that only complementary molecules which match very well canhybridize with one another. Low stringency enables molecules also topartially hybridize with relatively large sections of unpaired ormispaired bases.

The hybridization conditions are to be understood as being stringent, inparticular, if the hybridization is carried out in an aqueous solutioncontaining 2×SSC at 68° C. for at least 2 hours, followed by washingfirst in 2×SSC/0.1% SDS at room temperature for 5 minutes, then in1×SSC/0.1% SDS at 68° C. for 1 hour and then in 0.2% SSC/0.1% SDS at 68°C. for another hour.

A 2×SSC, 1×SSC or 0.2×SSC solution is prepared by diluting a 20×SSCsolution appropriately. A 20×SSC solution contains 3 mol/l NaCl and 0.3mol/l Na citrate. The pH is 7.0. The skilled worker is familiar with themethods for hybridizations of polynucleotides under stringentconditions. Appropriate instructions can be found in specialist bookssuch as, in particular, Current Protocols in Molecular Biology (WileyInterscience; editors: Frederich M. Ausubel, Roger Brant, Robert E.Kingston, David J. Moore, J. G. Seidmann, Kevin Struhl; ISBN:0-471-50338-X).

The yeast vectors can be divided into different subgroups. YIp vectors(yeast integrating plasmids) essentially correspond to the vectors usedin bacteria for clonings, but contain a selectable yeast gene (e.g.URA3, LEU2).

Only when the foreign DNA integrates into a yeast chromosome afterintroduction of said vector, are these sequences replicated togetherwith the chromosome and, with the formation of a clone, stablytransferred to all daughter cells.

Based on this method, plasmids have been derived which can replicateautonomously owing to eukaryotic ORIs (origins of replication). Suchyeast vectors are referred to as YRp vectors (yeast replicatingplasmids) or ARS vectors (autonomously replicating sequence).

Furthermore, there are YEp vectors (yeast episomal plasmids) which arederived from the yeast 2 μm plasmid and which contain a selective markergene. The class of the YAC vectors (yeast artificial chromosome) behavelike independent chromosomes.

A yeast vector containing a gene to be expressed is introduced into theyeast by means of transformation in order for said gene to be able to beexpressed. Examples of methods suitable for this purpose areelectroporation or incubation of competent cells with vector DNA.Suitable yeast expression promoters are known to the skilled worker,examples being the SOD1 promotor (superoxide dismutase), ADH promotor(alcohol dehydrogenase), the promotor of the gene for acidicphosphatase, HXT2 promotor (glucose transporter 2), HXT7 promotor(glucose transporter 7), GAL2 promotor (galactose transporter) andothers. The construct comprising a yeast expression promotor and a geneto be expressed (e.g. GLUT4V85M) is, for the purpose of expression, partof a yeast vector. To carry out expression, said yeast vector may be aself-replicating particle which is independent of the yeast genome ormay be stably integrated into the yeast genome. A suitable yeast vectoris in principle any polynucleotide sequence which can be propagated in ayeast. Yeast vectors which may be used are in particular yeast plasmidsor yeast artificial chromosomes. Yeast vectors usually contain an originof replication (2μ, ars) or starting the replication process and aselection marker which usually comprises an auxotrophy marker or anantibiotic resistance gene. Examples of yeast vectors known to theskilled worker are pBM272, pCS19, pEMBCYe23, pFL26, pG6, pNN414, pTV3,p426MET25, p4H7 and others.

In accordance with the present invention, selection of a cell means thespecific concentration thereof, owing to a selection marker such as, forexample, resistance to an antibiotic or the ability to grow on aparticular minimal medium, and furthermore the isolation and subsequentcultivation thereof on an agar plate or in submerged culture.

Cultivation, transformation and selection of a transformed yeast celland also expression of a protein in a yeast cell are among the methodscommonly used by the skilled worker. Instructions regarding said methodscan be found in standard text books, for example in Walker Graeme M.:Yeast Physiology and Biotechnology, Wiley and Sons, ISBN: 0-471-9446-8or in Protein Synthesis and Targeting in Yeast, Ed. Alistair J. P.Brown, Mick F. Fruite and John E. G. Mc Cartly; Springer Berlin; ISBN:3-540-56521-3 or in “Methods in Yeast Genetics, 1997: A Cold SpringHarbor Laboratory Course Manual; Adams Alison (Edt.); Cold Spring HarborLaboratory; ISBN: 0-87969-508-0”.

The yeast Saccharomyces cerevisiae has 17 known hexose transporters andadditionally three known maltose transporters, which are capable oftransporting hexoses into said yeast, provided that their expression issufficiently high. In one known strain all transporters suitable forhexose uptake were removed by deletion. Said strain contains merely justthe two genes MPH2 and MPH3 which are homologous to maltose transportproteins. The two genes MPH2 and MPH3 are repressed in the presence ofglucose in the medium. Wieczorke et al., FEBS Lett. 464, 123-128 (1999)describe the preparation and characterization of this yeast strain. Saidstrain is not able to propagate on a substrate containing glucose assole carbon source. It is possible to select from said strain mutantswhich functionally express GLUT1, starting from a corresponding vector(hxt fgy1-1 strain).

If the yeast strain hxt fgy1-1 is transformed with a plasmid vectorwhich carries a GLUT4 gene under control of a yeast promotor, still onlyvery little glucose is transported. Functional GLUT4 expression requiresfurther adjustments to this yeast strain in order to make possible asignificant glucose transport by means of GLUT4. Such yeast strainswhose cells take up glucose by means of a single glucose transporterGLUT4 can be isolated on substrates having glucose as sole carbonsource. For this purpose, a yeast hxt fgy1-1 strain carrying a GLUT4gene under the functional control of a yeast promotor is transformed.These yeast cells transformed in this way are applied to a nutrientmedium containing glucose as sole carbon source and are incubatedthereon. After a few days of incubation at, for example 30° C., thegrowth of individual colonies is observed. One of these colonies isisolated. The removal of the yeast plasmid from said colony preventspropagation on the nutrient medium containing glucose as sole carbonsource. If this strain which no longer contains a vector plasmid isagain transformed with a yeast vector carrying a GLUT4 gene under thefunctional control of a yeast promotor, said strain is then again ableto propagate on a medium containing glucose as sole carbon source.

The abovementioned yeast strains are the subject matter of InternationalApplication PCT/EP02/01373, filed on Feb. 9, 2002, which claims thepriority of DE 10106718.6 of Feb. 14, 2002.

Yeast strains whose indigenous transporters for hexoses (glucosetransporters) in their entirety are no longer functional have alreadybeen deposited at an earlier date in connection with InternationalApplication PCT/EP02/01373 with the Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSMZ) under the number DSM 14035,DSM 14036 or DSM 14037.

The polynucleotide and amino acid sequences of GLUT4 are accessible, forexample, via the following entries in gene banks M20747 (cDNA; human),EMBL: D28561 (cDNA; rat), EMBL: M23382 (cDNA; mouse), Swissprot: P14672(protein; human), Swissprot: P19357 (protein; rat) and Swissprot: P14142(protein; mouse).

Polynucleotide sequences and amino acid sequences of GLUT1 are disclosedunder the following code numbers of the databases indicated: EMBL:M20653 (cDNA; human), EMBL: M13979 (cDNA; rat), EMBL: M23384 (cDNA;mouse), Swissprot: P11166 (protein; human), Swissprot: P11167 (protein;rat) and Swissprot: P17809 (protein; mouse).

Pharmaceuticals are dosage forms of pharmacologically active substancesfor the therapy of diseases or bodily malfunctions in humans andanimals. Examples of dosage forms for oral therapy are powders,granules, tablets, pills, lozenges, sugar-coated tablets, capsules,liquid extracts, tinctures and syrups. Examples which are used forexternal application are aerosols, sprays, gels, ointments or powders.Injectable or infusible solutions allow parenteral administration, usingvials, bottles or prefilled syringes. These and other pharmaceuticalsare known to the skilled worker in the field of pharmaceuticaltechnology.

Excipients for formulating a pharmaceutical made possible thepreparation of the active substance with the purpose of optimizing theapplication, distribution and development of action of the activeingredient for the particular application. Examples of such excipientsare fillers, binders, disintegrants or glidants, such as lactose,sucrose, mannitol, sorbitol, cellulose, starch, dicalcium phosphate,polyglycols, alginates, polyvinylpyrrolidone, carboxymethylcellulose,talc or silicon dioxide.

Diabetes manifests itself by the excretion of glucose together with theurine with an abnormal increase in the blood glucose level(hyperglycaemia), owing to a chronic metabolic condition due to insulindeficiency or reduced insulin action. The lack of, or reduced, insulinaction leads to insufficient absorption and conversion by the cells ofthe glucose taken up into the blood. In fatty tissue,insulin-antagonistic hormones have the effect of increasing lypolysisaccompanied by an increase in the free fatty acid levels in the blood.

Adiposity (obesity) is the abnormal weight gain owing to an energyimbalance due to excessive intake of calories, which constitutes ahealth risk.

The amount of a hexose which is taken up by a provided yeast strain asdescribed just above can be determined by means of uptake studies usingradioactively labeled glucose. For this purpose, a particular amount ofthe yeast cells is suspended in, for example, 100 μl of a buffer, forexample at a concentration of 60 mg (wet weight) per ml, and admixedwith a defined amount of ¹⁴C- or ³H-labeled glucose as sole carbonsource. The cells are incubated, and defined amounts thereof are removedat specific times. The amount of glucose taken up is determined with theaid of LSC (Liquid Scintillation Counting). The amount of a hexose whichis taken up by a yeast strain provided and as just described above may,however, also be determined by means of a growth assay on mediacontaining glucose as sole carbon source. For this purpose, the rate ofgrowth of the strain is determined, after addition of the compound, forexample by measuring the optical density of the culture at 600 nm atregular intervals, and this value is compared with the rate of growth ofa control strain (e.g. yeast wild-type strain).

A compound is provided, in particular, by chemical synthesis or byisolating chemical substances from biological organisms. It is alsopossible to carry out chemical synthesis in an automated manner. Thecompounds obtained by synthesis or isolation can be dissolved in asuitable solvent. Suitable solvents are in particular aqueous solutionswhich contain a specific proportion of an organic solvent such as, forexample, DMSO (dimethylsulfoxide).

Conducting a strain of the yeast with a compound for identifying acompound in accordance with an invention mentioned above is done inparticular in automated laboratory systems provided therefor. Suchsystems may comprise specifically prepared chambers with depressions, ormicrotiter plates, Eppendorf tubes or laboratory glassware. Automatedlaboratory systems are usually designed for high throughput rates. Amethod such as the one mentioned above, carried out with the aid of anautomated laboratory system, is therefore also referred to as HTS (HighThroughput Screening).

Seq ID No. 1 discloses a polynucleotide sequence comprising the codingregion of the GLUT4V85M protein. Seq ID No. 2 discloses the amino acidsequence of the GLUT4V85M protein. Seq ID No. 3 discloses thepolynucleotide sequence of the p4H7GLUT4V85M vector.

EXAMPLES Use of Yeast Strains

All of the yeast strains described herein were derived from strainCEN-PK2-1C (MATa leu2-3, 112 ura3-52 trp1-289 his3-Δ1MAL2-8^(c) SUC2).The preparation of a yeast strain having deletions in the hexosetransporter genes (HXT) has been described by Wieczorke et al., FEBSLett. 464, 123-128 (1999): EBY-18ga (MATa Δhxt1-17 Δgal2 Δagt1 Δstl1leu2-3, 112 ura3-52 trp1-289 his3-Δ1 MAL2-8^(c) SUC2), EBY.VW4000 (MATaΔhxt1-17 Δgal2 Δagt1 Δmph2 Δmph3 Δstl1 leu2-3, 112 ura3-52 trp1-289his3-Δ1 MAL2-8^(c) SUC2). The media were based on 1% yeast extract and2% peptone (YP), while the minimal media were composed of 0.67% Difcoyeast nitrogen base without amino acids (YNB) and contained additivesrequired for auxotrophy and different carbon sources. The yeast cellswere grown under aerobic conditions at 30° C. on a rotary shaker or onagar plates. Cell growth was monitored by measuring the optical densityat 600 nm (OD₆₀₀) or by determining the diameter of yeast colonies.

Determination of Glucose Uptake

Glucose transport was measured as uptake of D-[U-¹⁴C]-glucoses(Amersham) and the kinetic parameters were determined from Eadie-Hofsteeplots. The cells were removed by centrifugation, washed withphosphate-buffer and resuspended in phosphate buffer at a concentrationof 60 mg (wet weight) per ml. Glucose uptake was determined for glucoseconcentrations between 0.2 and 100 mM, and the specific activity of thesubstrate was between 0.1 and 55.5 kBq μmol⁻¹. The cells and the glucosesolutions were preincubated at 30° C. for 5 minutes. Glucose uptake wasstarted by adding radioactive glucose to the cells. After incubation for5 seconds, 10 ml of ice-cold stop buffer (0.1 M KiPO₄, pH 6.5, 500 mMglucose) were added and the cells were removed quickly by filtering onglass fiber filters (Ø=24 mm, Whatman). The filters were quickly washedthree times with ice-cold buffer and the radioactivity incorporated wasmeasured using a liquid scintillation counter. An addition bycytochalasin B (final concentration 20 μM, dissolved in ethanol) wasmeasured in a 15-second uptake assay with 50 mM or 100 mM radioactiveglucose, after the cells had been incubated in the presence of theinhibitor or of only the solvent for 15 minutes.

A novel heterologous expression system for glucose transporters frommammalian cells has been developed. The system is based on an S.cerevisiae strain from which all endogenous glucose transporters havebeen removed destroying the encoding genes. Said strain is no longerable to take up glucose via the plasma membrane and to grow with glucoseas sole carbon source. In order to integrate the heterologous glucosetransporters of humans or of other mammals, GLUT1 and GLUT4 in an activeform into the yeast plasma membrane, additional mutations had to beintroduced into the yeast strain. GLUT1 is active only in an fgy1-1mutant strain and GLUT4 only in fgy1-1 fgy4-X double mutants. The FGY1gene has been cloned. It is the S. cerevisiae ORF YMR212c. With respectto the function, the results indicate that either Fgy1 or a productgenerated by Fgy1 inhibits the activity of human glucose transporters oris involved in fusing the GLUT-transporting vesicles to the plasmamembrane.

In contrast to GLUT1 and similarly to mammalian cells, a largeproportion of the GLUT4 proteins in the yeast is located inintracellular structures. A total of nine recessive mutants wereisolated (fgy4-1 to fgy4-9) in which GLUT4 is now directed further tothe plasma membrane and, in the case of a concomitant fgy1-1 mutation,becomes active there.

The insertion gene bank described by Bruns et al. (Genes Dev. 1994; 8:1087-105) was used for complementation analysis. The hxt fgy1-1 strainwas transformed first with a GLUT4 plasmid and then with the mobilizedinsertion gene bank. This was followed by screening for transformantswhich were able to grow on glucose medium. It turned out that in one ofthe mutants studied the ERG4 gene had been destroyed. ERG4 codes for anenzyme (oxidoreductase) of ergosterol biosynthesis. This enzyme, sterolC-24(28)-reductase catalyzes the last step of ergosterolbiosynthesis andconverts ergosta-5,7,22,24,(28)-tetraenol to the final productergosterol. The Erg4 protein presently contains eight transmembranedomains and is located in the endoplasmic reticulum. An erg4 mutant isviable, since incorporation of the ergosterol precursors into the yeastmembranes compensates for the loss of ergosterol.

The inhibiting influence of Erg4 on GLUT4 functionality was confirmed byspecific deletion of erg4 in the hxt fgy1-1 strain. The resulting strain(hxt fgy1-1 Δerg4) was referred to as SDY022.

Protein interaction assays with the aid of the split-ubiquitin systemshowed that human GLUT4 directly interacts with yeast Erg4. It cantherefore be assumed that the yeast Erg4 protein in the endoplasmicreticulum either directly prevents further translocation of GLUT4 ormodifies GLUT4 in some way which is important for translocation and/orfunction.

Likewise, it was shown that deletion of ERG4 in the hxt null strainalone, i.e. despite functional FGY1, activates GLUT1, but not GLUT4. Theresults of the growth assay are summarized in Table 1.

In order to rule out that Ergosterol itself exerts a negative influenceon GLUT4, growth assays were carried out on agar plates containingErgosterol under aerobic conditions. Any yeast strains transformed withGLUT4 were unable to grow under these conditions (Table 2). The GLUT1transformants in the hxt fgy1-1 strain showed, in contrast to aerobicgrowth, no growth on glucose under anaerobic conditions. GLUT1transformants were able to grow only after deletion of ERG4.

The exchange of Val85 for Met by in vitro mutagenesis rendered GLUT4independent of the fgy1-1 mutation and resulted in GLUT4V85M beingfunctional even in an hxt erg4 strain.

This observation indicates that Fgy1 acts directly or indirectly on thisposition which is located within the second transmembrane helix of GLUTtransporters.

Table 3 displays the descriptions of the yeast strains deposited inconnection with the present patent application with the DeutscheSammlung von Mikroorganismen and Zellkulturen (DSMZ)-Mascheroder Weg 1b38124 Brunswick, Germany.

TABLE 1 Growth of GLUT1 and GLUT4 transformants on glucose medium.Genotype 1% Glucose 1% Glucose Δhxt fgy1-1 GLUT4 − GLUT1 ++ Δhxt fgy1-1Δerg4 GLUT4 ++ GLUT1 ++ Δhxt fgy1-1 Δerg4 Vector − Vector − Δhxt fgy1-1Δerg5 GLUT4 − GLUT1 ++ Δhxt fgy1-1 Δerg4 Δerg5 GLUT4 + GLUT1 ++ ΔhxtΔerg4 GLUT4 − GLUT1 + Δhxt Δerg5 GLUT4 − GLUT1 −

TABLE 2 Growth of GLUT1 and GLUT4 transformants on glucose medium withor without ergosterol under anaerobic conditions 1% Glucose + Genotype1% Glucose 33 mg/l Ergosterol Δhxt fgy1-1 GLUT1 − − GLUT4 − − Δhxtfgy1-1 Δerg4 GLUT1 − ++ GLUT4 − − Δhxt fgy1-1 Δerg5 GLUT1 − − GLUT4 − −Δhxt fgy1-1 Δerg4 Δerg5 GLUT1 − ++ GLUT4 − − Δhxt Δerg4 GLUT1 − (+)GLUT4 − − Δhxt Δerg5 GLUT1 − − GLUT4 − −

TABLE 3 Features of the deposited yeast strains (Saccharomycescerevisiae) Number of deposit with the DSMZ Genotype Phenotype PlasmidDSM 15187 MATa Δhxt1-17 Δgal2 Strain growth with — Δagt1 Δstl1 Δmph2 1%maltose as Δmph3 Δerg4 leu2-3, 112 carbon source; ura3-52 trp1-289his3-Δ1 auxotrophic for MAL2-8^(c) SUC2 glucose, leucine, tryptophan,histidine and uracil DSM 15184 MATa Δhxt1-17 Δgal2 Strain growth with —Δagt1 Δstl1 Δerg4 fgy1-1 1% maltose as leu2-3, 112 ura3-52 trp1- carbonsource; 289 his3-Δ1 MAL2-8^(c) auxotrophic for SUC2 glucose, leucine,tryptophan, histidine and uracil DSM 15185 MATa Δhxt1-17 Δgal2 Straingrowth with p4H7GLUT4V85M Δagt1 Δstl1 Δmph2 1% maltose as (Selectionmarker Δmph3 Δerg4 leu2-3, 112 carbon source; URA3), = ura3-52 trp1-289his3-Δ1 auxotrophic for Seq ID No. 3 MAL2-8^(c) SUC2 glucose, leucine,tryptophan and histidine DSM 15186 MATa Δhxt1-17 Δgal2 Strain growthwith p4H7GLUT4V85M Δagt1 Δstl1 Δerg4 fgy1-1 1% maltose as (Selectionmarker leu2-3, 112 ura3-52 trp1- carbon source; URA3) = 289 his3-Δ1MAL2-8^(c) auxotrophic for Seq ID No. 3 SUC2 glucose, leucine,tryptophan and histidine DSM 15188 MATa Δhxt1-17 Δgal2 Strain growthwith p4H7GLUT4V85M Δagt1 Δstl1 Δmph2 1% maltose as (Selection markerΔmph3 leu2-3, 112 ura3- carbon source; URA3) = 52 trp1-289 his3-Δ1auxotrophic for Seq ID No. 3 MAL2-8^(c) SUC2 glucose, leucine,tryptophan and histidine

Basic medium: 0.67% Yeast Nitrogen Base without amino acids (Difco); pH6.2. Auxotrophy supplementation: Leucine (0.44 mM), tryptophan (0.19mM), histidine (0.25 mM9, uracil (0.44 mM). Maltose may be between 1-2%.

1. An engineered strain of Saccharomyces cerevisiae (a) which can nolonger grow on substrates with hexoses as the only carbon source, (b)which lacks a functional protein expressed from Saccharomyces cerevisiaeopen reading frame YMR212c (Fgy1) protein, and (c) which further lacks afunctional sterol C-24(28)-reductase (Erg4) protein, whose ability ofgrowing on a substrate with a hexose as the only carbon source isrestored when a glucose transporter type 4 (GLUT4) gene is expressed inthis strain, wherein the GLUT4 gene encodes an amino acid sequencecomprising SEQ ID NO:2, wherein the methionine residue of position 85 isa valine residue.
 2. The strain of claim 1, wherein said GLUT4 generestores said strain's ability to grow on a substrate with a hexose asthe only carbon source.
 3. The strain of claim 2, wherein said GLUT4gene is a recombinant GLUT4 gene which is under the functional controlof a promoter which can be expressed in yeast.